Purpose:
The inwardly-rectifying potassium channel Kir7.1 is present in the apical processes of retinal pigment epithelial (RPE) cells. Several mutations in the gene that encodes Kir7.1 (KCNJ13) cause blindness in the allelic disorders of Snowflake Vitreoretinal Degeneration (SVD) and Lebers Congenital Amaurosis (LCA16). In this study, we treated two Kir7.1 nonsense mutations that result in LCA16 (W53X and R166X) with the read-through compounds Ataluren (PTC-124; AdooQ Biosciences) and a novel small molecule, RTC-14.

Methods:
Chinese Hamster Ovary (CHO-K1) cells were transfected with N-terminal GFP-fused W53X and R166X mutant plasmids. The cells were then treated with two different concentrations, 5 µM and 10 µM, of the read-through compounds PTC-124 or RTC-14 after eight hours of transfection, and the cells were incubated with these drugs for 36 hours. Whole-cell patch clamp electrophysiology was performed on the transfected cells. Function of the Kir7.1 channel was measured in the presence of Cs+ to block function, or the highly permeant Rb+ to enhance current. The students T-test was used and significance was determined at the P<0.05 level.

Results:
Both W53X and R166X transfection resulted in non-measurable Kir7.1 current as compared to the wild-type Kir7.1 channel, and the cells were depolarized. Upon treatment of cells with PTC-124 we detected no measurable difference in either the current amplitude or the resting membrane potential (Vm). In contrast, RTC-14 showed partial rescue of both the current amplitude and Vm. R166X expressing cells responded to RTC-14 treatment by a 15 mV hyperpolarizing shift in Vm (-25.92 ± 2.7 to -40.16 ± 4.05 mV; P<0.02) and an increase in current amplitude measured at -150 mV from -98.5 ± 24.7 to -122.8 ± 28.15 pA, P = 0.52. W53X, however, showed a 33 mV hyperpolarization shift in Vm (-29.37 ± 3.55 to -61.94 ± 2.9 mV; P<0.001). We also noticed a four-fold augmentation in both inward (-80.4 ± 11.2 to -316.16 ± 60.8 pA at -150 mV; P<0.01) and outward (-7.4 ± 2.4 to 33.3 ± 11 pA at -40 mV; P<0.02) current amplitude.

Conclusions:
RTC-14 is effective in the abrogation of both Vm and current amplitude in the LCA16 nonsense mutations W53X and R166X. W53X was more amenable to read-through by RTC14 as compared to R166X. Our studies clearly demonstrate the potential of the small-molecule RTC-14 as a therapeutic agent for treating LCA16 due to Kir7.1 channel nonsense mutations.